Leptin is a hormone that controls fat storage and energy expenditure. Leptin is made by adipocytes and acts on hypothalamic neurons that regulate energy balance. High leptin levels are an indicator of sufficient energy stores and should cause cessation of eating. However, most obese humans are leptin resistant. Leptin interacts with a JAK/STAT pathway-coupled receptor, and leptin resistance can be caused by downregulation of the pathway downstream of the receptor. If additional regulators of leptin receptor signaling in hypothalamic neurons could be identified, manipulation of their activities might provide ways to overcome leptin resistance. This proposal describes a new way to find such regulators using Drosophila genetics. Drosophila has dedicated adipocytes, and the activities of specific sets of brain neurons control fat storage and metabolism. The preliminary results reported here show that Drosophila fat content is regulated by a leptin-like JAK/STAT signaling pathway that acts in fat-regulating 'Fru' neurons. These findings show that flies and mammals use some similar genetic and neural mechanisms for control of fat storage. Drosophila is a model system that is amenable to fast and inexpensive forward genetic screening, and our results suggest that conserved JAK/STAT regulators identified in Drosophila would have relevance for research in mammalian systems. The objective of the first specific aim is to find proteins that affect fat content by regulating JAK/STAT signaling in Fru neurons. These will be identified by screening a set of ~200 genes identified as modulators of the JAK/STAT pathway in cultured cells, about 75% of which have human orthologs or relatives. Expression of each gene will be knocked down in Fru neurons using transgenic RNAi, and those genes for which knockdown affects triglyceride levels will be selected for further study. The identified candidate genes will be prioritized based on sequence relationships with human genes, effect size and direction, and availability of viable loss-of- function mutations and inserted elements that can be used for tagging. The objective of the second specific aim is to place the highest-priority subset of the regulators identified in Specific Aim 1 into their molecular and cellular contexts. Analysis of genetic epistasis will be used to determine whether the regulators act upstream or downstream of the receptor, and if they control the firing of Fru neurons. Regulators will be fluorescently tagged in vivo to reveal their cellular and subcellular expression patterns. The same tagging strategy will be used to create 'driver' lines that will confer gene expression in neurons that normally express the regulator, allowing manipulation of their activities. The expected outcome of the research proposed in specific aims 1 and 2 is the definition of a set of conserved modulators of JAK/STAT signaling that function downstream of the receptor in fat-regulating neurons. Negative regulators identified in these experiments might be potential drug targets whose inhibition could upregulate anorexigenic leptin signaling in leptin-resistant obese individuals.